Assay for the proteolytic activity of serotype a from clostridium botulinum

ABSTRACT

A label-based assay is described, through modifications of substrate  strure and derivatization of serum albumin, which can be used to determine type A proteolytic activity without separation of products.

INTRODUCTION

Type A botulinum neurotoxin (botox A) is one of seven serologicallydistinct neurotoxins produced by various strains of the anaerobic, sporeforming bacterium, Clostridium botulinum. Together with the structurallyrelated neurotoxin from Clostridium tetani, they are among the mostpotent toxins known [Eisel et al. (1986) EMBO J. 5: 2495-2502; Nieman(1991) In: Sourcebook of Bacterial Protein Toxins (J. Alouf and J Freer,Eds.) pp. 303-348, Academic Press, New York; Simpson (1981) Pharmacol.Rev. 33: 155-188; Dolly (1992) In: Handbook of Experimental Pharmacology(H. Herken and F. Hucho, Eds.), pp. 681-717. Springer-Verlag, Berlin.]Nonetheless, these toxins have proven to be highly useful tools forresearch on the mechanisms of neurotransmitter release [Nieman (1991)Trends Cell Biol. 4: 179-185; Schiavo et al., (1994) Cell Biol. 5:221-229] and have even been used as drugs in humans, to treat certaintypes of muscle dysfunctions [Jancovic and Brin (1992) New Engl. J. Med.324: 1186-1194]. All references cited herein supra and infra are herebyincorporated in their entirety by reference thereto.

Recognition of a consensus zinc-binding motif, HEXXH, in each of thebotulinum and tetanus toxins was soon followed by reports that thesetoxins were zinc endoproteinases, highly specific for intracellularproteins involved in neurotransmission [Jongeneel et al. (1989) FEBSLett. 242: 211-214; Schiavo et al. (1992b) EMBO J. 11, 3577-3583;Schiavo et al. (1992a) Nature 359: 832-835; Schiavo et al. (1993) J.Biol. Chem. 268: 23784-23787; Blasi et al. (1993) Nature 365: 160-163;Yamasaki et al. (1994) J. Biol. Chem. 269: 12764-12772]. For example,botox B and tetanus neurotoxins both cleave the same peptide bond in thesynaptic vesicle protein synaptobrevin (also called VAMP), while botox Aand botox E both cleave the synaptosomal protein SNAP-25, albeit atdifferent sites (Schiavo et al., 1992a, ibid., 1993, ibid.; Blasi etal., 1993, ibid.). Proteolytic cleavage incapacitates these proteins,preventing neurotransmitter release. Toxicity is therefore a consequenceof clostridial neurotoxin endoproteinase activity.

In earlier work [Schmidt and Bostian, (1995) J. Prot. Chem. 14:703-708], we found that a 17-amino acid peptide, corresponding toresidues 187-203 of SNAP-25 [Oyler, et al. ,(1989) J. Cell Biol. 109:3039-3052], could serve as a good substrate for the proteolytic activityof botox A. The peptide was cleaved by botox A at a singleglutaminyl-arginine bond, corresponding to residues 197 and 198 ofSNAP-25, confirming earlier reports on the enzymatic specificity ofbotox A in synaptosomal preparations. Extending the peptide to thecarboxy-terminus of SNAP-25 (187-206) or including up to 40 residues(167-206) had no positive influence on the rate of hydrolysis, comparedto that with the 17-residue peptide. Others have reported that tetanusneurotoxin and types B, D, and F botulinum neurotoxins hydrolyzesynthetic peptides, but relatively large peptides (>34 residues) wererequired [Shone et al., (1993) Eur. J. Biochem. 217: 965-971; Foran etal., (1994) Biochemistry 33: 15365-15374; Yamasaki et al., 1994, ibid.;Cornille et al., (1994) Eur. J. Biochem. 222: 173-181; Shone andRoberts, (1994) Eur. J. Biochem. 225: 263-270].

Type A botulinum toxin is currently employed as a drug to treat avariety of human muscle dysfunction. Current methods for estimatingbotulinum neurotoxin concentrations are the mouse lethality assay[Siegel and Metzger (1979) Appl. Environ. Microbiol. 38: 606-611], andan antibody neutralization test [Siegel (1988) J. Clin. Microbiol. 26:2351-2356]. Both require the use of animals, can take up to four days tocomplete, and are inherently inaccurate. Dosages are calculated on thebasis of the mouse lethality bioassay. However, this is unsatisfactory,because both assays are lengthy, inherently inaccurate, and require theuse of animals. Furthermore, different results can be obtained dependingon which protocol was used. Comparison of different lots of toxin, ortoxin prepared in different laboratories, is difficult. Since theclostridial neurotoxins are enzymes, it follows that preparations couldbe quantitated as with any other enzyme, by determining the rate of thereaction catalyzed by the preparation. For botox A, this can be done byHPLC separation and quantitation of substrate hydrolysis products[Schmidt and Bostian (1995) J. Prot. Chem. 14: 703-708].

In this application, we describe an enzymatic assay for the quantitationof type A botulinum toxin wherein botox A can be convenientlyquantitated, standardized, and compared on the basis of specificenzymatic activities, as is commonly done for virtually all otherenzymes. The assay does not require the use of animals and can be donein one hour or less since no separation of hydrolysis products isneeded.

SUMMARY

The present invention relates to botox A substrate peptides and theirstructural requirements and modifications suitable for the determinationof type A botulinum toxin enzymatic (proteolytic) activity in alabel-based assay. The present assay allows, for the first time,different preparations of type A botulinum toxin to be convenientlyquantitated, standardized, and compared on the basis of specificenzymatic activities.

After toxin-catalyzed hydrolysis of the peptide substrates, results arequantitated by the addition of a label, followed by measuring the amountof label. Assays are very sensitive (2 to 5 nanograms per millilitertoxin can be detected), are highly accurate (typical standard deviationsof triplicate determinations are less than 5%), do not require priorseparation of products, can be completed in one hour, and do not requirethe use of animals.

In contrast, the conventional method for estimating concentrations ofbotulinum toxins is the mouse lethality bioassay. This method is notuniversally standardized, can take up to four days to complete, ishighly inaccurate, and obviously requires the use of animals. Since thelethality of botulinum toxin is a consequence of its proteolyticactivity, the enzymatic toxin assay can replace the mouse lethalitybioassay in many applications.

Therefore, it is one object of the present invention to providesubstrate peptides for use in an assay for the determination of type Abotulinum toxin enzymatic activity.

It is another object of the present invention to provide a method fordetecting and measuring type A botulinum toxin enzymatic activity ormeasuring the lethality of type A botulinum in a sample. In addition,the method can be used for standardization of different lots of toxin,and tracking of toxin during production and purification of drugs orsolutions which contain type A botulinum toxin.

It is still a further object of the present invention to provide amethod for screening compounds for type A botulinum inhibitory orstimulatory activity. The enzymatic assay described herein can be easilyadapted to screen hundreds of compounds at once for toxin-inhibitoryactivity which can be further tested for effectiveness as anti-toxindrugs for treating a person with botulinum intoxication. Stimulatorycompounds can be tested as drugs for treating an ever-expanding numberof human muscle dysfunctions.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and accompanying drawing where

FIG. 1 represents the hydrolysis of 1 mM peptide 5, catalyzed by botoxA. At the indicate times, aliquots of 20 μl were removed and reactedwith fluorescamine, as described in the Materials and Methods below.

DETAILED DESCRIPTION

The present invention relates partially to peptides for use in an assayfor the determination of type A botulinum toxin enzymatic (proteolytic)activity in a sample. Samples include, but are not limited to raw,cooked, or processed foods, beverages, animal feed, soil and watersamples, pond sediments, lotions, and cosmetics, clinical drugs andsolutions.

The assay of the present invention is based on the well-known fact thatproteolytic cleavage of peptides or proteins results in production ofnew free amino groups. In this assay, type A botulinum toxin cleavesonly one bond in the peptide substrate; therefore, one mole of freeamino group results from each mole of peptide substrate that ishydrolyzed. The extent or rate of hydrolysis can be measured bydetermining the amount of, or rate of appearance of, free amino group.The concentration of toxin in an unknown sample can then be calculated,based on measurements of the extent or rate of hydrolysis effected byknown concentrations of toxin.

In principle, any reagent that reacts with amino groups could be used asa label for this determination. However, in most cases the absorbancespectra of reactants and products are similar or identical. Therefore, ameans of separating and identifying reactants and products must bedeveloped and applied. Examples of reagents in this category are phenylisothiocyanate and 2,4,6-trinitrobenzene-1-sulfonic acid.

Colorimetric reagents are commonly considered to be those which have nocolor, but which react with a certain substance or type of substance togive a colored product that is visible to the human eye. Quantitationinvolves measurement of the optical density of the solution with aspectrophotometer. For amino groups, a useful colorimetric reagent isninhydrin, which reacts with primary amines to give a purple color(secondary amines yield a yellow color). However, it also reacts withammonia, which is present in most solutions, unless great pains aretaken to exclude or remove it. Therefore, chromatographic separation ofproducts is necessary to avoid high background readings, which wouldcompromise sensitivity.

Fluorescent reagents that react with primary and secondary aminesinclude 5-dimethylaminonaphthalene-1-sulfonyl chloride,4-dimethylaminoazobenzene-4'-sulfonyl chloride, and4-N,N-dimethylaminoazobenzene 4'-isothiocyanate. However, these reagentsare fluorescent even before reaction with amino groups, necessitatingthe separation of products from unreacted reagents which is timeconsuming and requires expensive equipment.

Another commonly-used reagent for detection and quantitation of aminogroups is o-phthalaldehyde (OPA). This compound is not fluorescent, butforms fluorescent derivatives upon reaction with primary amines.Unfortunately, it also reacts strongly with ammonia, leading to highbackground readings.

If an assay for the proteolytic activity of type A botulinum toxinincludes steps designed to separate products before quantitation (forexample, see Schmidt and Bostian, 1995, ibid.), then most of theabove-mentioned reagents could be used. Resolution of products could beaccomplished by such techniques as high-pressure liquid chromatography(hplc), microbore or capillary liquid chromatography, or capillaryelectrophoresis. These add both time (methods development, validation,turnaround time, etc.) and significant expense (equipment purchase,maintenance, personnel training, etc.) to the procedure.

In the assay of the present invention, fluorescamine is used as thedetection reagent because: (a), it is not fluorescent; (b), it reactswith amino groups to give intensely fluorescent compounds; and (c), itdoes not react with ammonia. These properties have made fluorescaminethe reagent of choice in protease assays for many years. Nonetheless,fluorescamine will react with the native-sequence substrate peptide, dueto the presence of two amino groups. We have developed a set of peptidescontaining modified sequences which do not react with fluorescamine, butare good substrates for the proteolytic activity of type A botulinumneurotoxin. Thus, the requirement for expensive and time-consumingproduct separation has been completely eliminated. Only a relativelyinexpensive fluorimeter is needed.

In this context, in earlier work [Schmidt and Bostian (1995) ibid.], wefound that a 17-amino acid peptide,

S N K T R I D E A N Q R A T K M L, (SEQ ID NO:1), corresponding toresidues 187-203 of SNAP-25 could serve as a good substrate for theproteolytic activity of botox A. However, this peptide is not suitablefor use in the assay system described herein, because it reacts withlabeling reagents to produce derivatives, and in the case of afluoregenic label, a fluorescent derivative can seriously compromise theassay. The same can be said for larger peptides based on the SNAP-25sequence, and for intact SNAP-25. This is due to the presence of thealpha-amino group of the N-terminal residue, and the epsilon-aminogroups of the lysine residues at positions 3 and 15 in the sequence.This very high "background" fluorescence must be subtracted from thatobtained in an assay, which severely compromises the sensitivity.Alternatively, the assay components and products must be separated fromone another before quantitation, as described above, by procedures whichare lengthy and require the use of very expensive, and specializedequipment.

Two changes were introduced to reduce or eliminate the reactivity ofintact substrate with labeling or detection reagents: (1) acetylation ofthe alpha-amino group of the N-terminal residue; and (2) replacement ofboth lysines with arginines. With these changes, the "background" signalis typically less than 10% of the total in an assay. Therefore, it isnot necessary to separate assay components and products beforequantitation.

The amino acid sequences of the substrate peptides described in thisinvention are given below. In each case, the N-terminal amino group isacetylated, and the C-terminus is a carboxamide instead of a freecarboxy group. This improves substrate properties; that is, they arehydrolyzed by type A toxin more rapidly than substrate peptides withfree COOH at the C-termini. (see Schmidt and Bostian, 1995, ibid.).Acetylation of amino-terminal residues is a common practice in syntheticpeptide chemistry. In our case, it was done to eliminate a free aminogroup that would otherwise react with fluorescamine. Some of thesubstrate peptides of the present invention are as follows:

    ______________________________________                                         1. S N R T R I D E A N Q R A T R M L (SEQ ID NO:2)                            2. S N R T R I D Q A N Q R A T R M L (SEQ ID NO:3)                            3. S N R B R I D E A N Q R A T R M L (SEQ ID NO:4)                            4. S N R T R I D E A N Q R A B R M L (SEQ ID NO:5)                            5. S N R T R I D E A N Q R A T R X L (SEQ ID NO:6)                            6. S N R B R I D E A N Q R A T R M (SEQ ID NO:7)                              7. S N R B R I D Q A N Q R A T R M L (SEQ ID NO:8)                            8. S N R B R I D Q A N Q R A T R M (SEQ ID NO:9)                              9. S N R B R I D B A N Q R A T R M L (SEQ ID                                                                         NO:10)                                10. S N R B R I D B A N Q R A T R M (SEQ ID NO:11)                            11. S N R T R I D Q A N Q R A T R M (SEQ ID NO:12)                            12. S N R T R I D B A N Q R A T R M (SEQ ID NO:13)                            13. S N R T R I D B A N Q R A T R M L (SEQ ID                                                                        NO:14)                                 ______________________________________                                    

14. Any substrate peptide containing one or more of the residue changesshown in peptides 2 to 5, inclusive, with respect to peptide 1, and/ordeletion of the carboxy-terminal leucine residue, and/or addition ofmore amino acids to the carboxy-terminal end, and/or addition of moreamino acids to the amino-terminal end, and/or deletion of one or moreamino acids from the amino-terminal end. For example the peptide S N R BR I D Q A N Q R A B R X L (SEQ ID NO: 15) would include many of thechanges.

15. Any substrate peptide, for type A botulinum toxin, containingmodified lysine residues, such as N (epsilon)-acetyl-lysine.

16. Any peptide that will serve as a substrate for type A botulinumtoxin, but will not react with labeling reagents.

Abbreviations for the amino acids are:

    ______________________________________                                        A            Alanine                                                          B            2-Aminobutyric acid                                              D            Aspartic acid                                                    E            Glutamic acid                                                    I            Isoleucine                                                       K            Lysine                                                           L            Leucine                                                          M            Methionine                                                       N            Asparagine                                                       Q            Glutamine                                                        R            Arginine                                                         S            Serine                                                           T            Threonine                                                        X            2-Aminohexanoic acid (norleucine)                                ______________________________________                                    

All the peptides described above can be used in a proteolytic assay tomeasure type A botulinum enzyme. Such an assay is performed as follows.One of the substrate peptides of the present invention is mixed withtype A botulinum toxin. Typical concentrations are about 0.001 M peptideand about 0.01-1 microgram per ml toxin. The mixture also contains zincchloride or zinc acetate at about 0.0002-0.0005 M. Zinc ion is added,because without it, the rate of catalysis by type A toxin is very low.The range specified is that which is most effective. Lowerconcentrations are insufficient, while higher concentrations areinhibitory (see Schmidt and Bostian, 1995, ibid.). Other salts of zinccould be used, provided that they are soluble. It is also possible thatother divalent cations could be substituted for zinc.

Acetylated bovine serum albumin is present in the reaction solution atabout 0.5-2.0 milligrams per ml, and a reducing agent, preferablydithiothreitol, at about 0.002-0.01 M. Assay volumes are usually about15-60 microliters. The mixture is incubated at about 20-37° C. for asuitable period of time, usually about 5-60 minutes. Incubation (assay)endpoint is a function of the toxin (enzyme) concentration. In order tocalculate an accurate initial rate of hydrolysis, it is necessary tostop the reaction before more than about 25% of the substrate iscleaved. Alternatively, the extent of hydrolysis at multiple time pointscan be determined, and the initial rate of hydrolysis is then calculatedby extrapolation to zero- or near zero-time. These are standardprocedures in enzymology well known to a person with ordinary skill inthe art.

The reaction is stopped by addition of about 0.5-1.0 M sodium borate, pH9.0-9.5, containing about 0.01 M iodoacetamide or sodium iodoacetate. Inorder to maximize fluorescent yield, measurements are done at pH 9.0-9.5(common practice in fluorimetry). Sodium iodoacetate or iodoacetamide isadded to eliminate reducing agent (dithiothreitol) in the assay mixture.If this is not done, the latter will react very rapidly withfluorescamine, and prevent its reaction with amino groups. Otherpossibilities include 4-vinylpyridine and N-ethylmaleimide.

After about 30 minutes at room temperature (typically about 18-23° C.),a fluorigenic reagent or other labeling reagent is added, that reactswith free amino groups to produce or fluorescent or other detectablederivative. Fluorescamine can be used at about 0.5-1.0 milligrams per mlin dimethylformamide. This is added with immediate and vigorous mixing.The fluorescence yield is then measured in a fluorimeter.

Hydrolysis of these peptides, catalyzed by type A botulinum neurotoxin,occurs between the glutaminyl-arginyl peptide bond that is found in eachpeptide. Quantitation can be achieved by comparing the fluorescenceyield of the above-described assay with that obtained from knownconcentrations of a peptide representing the C-terminal product of thehydrolysis reaction. For example, if substrate peptide number 1 is usedin the assay, then the appropriate C-terminal product peptide is: RATRML(residues 12-17 of SEQ ID NO: 2). The objective is to correlatefluorescence readings with the extent of hydrolysis. That is, if thesubstrate peptide is incubated with type A toxin for a period of time,then the reaction mixture is processed as described, and a fluorescencereading of 1250 is obtained, how many moles of substrate were cleavedduring that time? Before this question can be answered, one must knowthe fluorescence yield per mole of C-terminal product. The simplest wayto determine this is to synthesize the C-terminal product, then reactknown quantities of it with fluorescamine in the same way that it's donein an assay. Then, the fluorescence yield per mole of product can becalculated. The reaction rate can be expressed in standard terms, suchas moles of substrate hydrolyzed (or products formed) per unit of timeper mole (or mass) of enzyme (toxin). Using known concentrations ofpurified toxin, the hydrolysis rates for the peptides, per unit oftoxin, can be calculated. These results can then be used to determineconcentrations of toxin in preparations where the latter is unknown.

In other words, since type A botulinum toxin is an enzyme (a protease),it follows that concentrations of toxin in samples can be determined bymeasuring the amount of enzyme activity present, after the specificactivity of the toxin is determined. For many enzymes, specific activityis expressed as micromoles of substrate transformed per minute permilligram of enzyme, or μmole/min/μg. Using the assay techniquesdescribed in this disclosure, the specific activity of type A botulinumtoxin can be obtained by measuring the rate of proteolysis of aparticular substrate peptide, catalyzed by a known concentration oftoxin. (Once the specific proteolytic activity of type A toxin isdetermined, it need not be re-determined each time an assay isperformed). Toxin concentration in a test sample can then be calculated,by determining the rate at which the test sample cleaves the samesubstrate. For example, the specific activity of type A toxin againstpeptide 5 (in Table II below) was 21 μmol/min/mg, when the initialsubstrate concentration was 1.0 mM. Therefore, if the rate of hydrolysisof 1.0 mM peptide 5, catalyzed by a sample containing an unknownconcentration of type A toxin in a total assay volume of 20 μL, isdetermined to be 0.00042 μmol/min, then the amount of enzyme (type Atoxin) present was: 0.00042/21=0.00002 mg, at a concentration of0.00002/0.02=0.001 mg/ml. Thus, the concentration of type A botulinumtoxin can be determined in preparations (such as, for example, thoseintended for human clinical use), without doing mouse lethality assays.

The assay could be used to search for inhibitors of type A botulinumproteolytic activity. Peptides that are slowly hydrolyzed, or not atall, might be starting points for drug development.

Based on information in Schmidt and Bostian, 1995 (ibid.), the minimumfunctional substrate is likely to be:

T R I D E A N Q R A T R M (SEQ ID NO:16)

or

R I D E A N Q R A T R M (SEQ ID NO:17)

The peptides listed below have been grouped on the basis of performanceas substrate and/or potential inhibitors. Included are all of thepeptides synthesized and tested, and many which have not beensynthesized. Categorization of the latter are based on experience andtheory. Note that many of the peptides contain lysine, rendering themless than ideal for a fluorimetric assay. If these are used assubstrates, then the best approach would be to separate the assayproducts by HPLC, microbore or capillary liquid chromatography, orcapillary electrophoresis.

Group 1, excellent substrates

    __________________________________________________________________________    1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17                             S  N  R  T  R  I  D  E  A  N  Q  R  A  T  R  M  L  SEQ ID NO:2                S  N  R  B  R  I  D  E  A  N  Q  R  A  T  R  M  L  SEQ ID NO:4                S  N  R  T  R  I  D  Q  A  N  Q  R  A  T  R  M  L  SEQ ID NO:3                S  N  R  T  R  I  D  B  A  N  Q  R  A  T  R  M  L  SEQ ID NO:14               S  N  R  B  R  I  D  Q  A  N  Q  R  A  T  R  M  L  SEQ ID NO:8                S  N  R  B  R  I  D  B  A  N  Q  R  A  T  R  M  L  SEQ ID NO:10               S  N  K  T  R  I  D  Q  A  N  Q  R  A  T  K  M  L  SEQ ID NO:18               S  N  K  B  R  I  D  E  A  N  Q  R  A  T  K  M  L  SEQ ID NO:19               S  N  K  T  R  I  D  B  A  N  Q  R  A  T  K  M  L  SEQ ID NO:20               S  N  K  B  R  I  D  Q  A  N  Q  R  A  T  K  M  L  SEQ ID NO:21               S  N  K  B  R  I  D  B  A  N  Q  R  A  T  K  M  L  SEQ ID                     __________________________________________________________________________    NO:22                                                                     

Any of the above peptides, with residue 6=L

Any of the above peptides, with residue 17 (L) deleted, with or withoutthe K/R substitutions described below

Any of the above peptides, with residue 3=K and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=K

Group 2, good substrates

    __________________________________________________________________________    1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17                             S  N  R  S  R  I  D  E  A  N  Q  R  A  T  R  M  L  SEQ ID NO:23               S  N  K  T  R  I  D  E  A  N  Q  R  A  T  K  M  L  SEQ ID NO:1                S  N  K  T  R  I  D  E  A  N  Q  R  A  T  K  X  L  SEQ ID NO:24               S  N  K  T  R  I  D  E  A  N  Q  R  A  B  K  M  L  SEQ ID NO:25               S  N  K  T  R  I  D  E  A  N  Q  R  A  C  K  M  L  SEQ ID NO:26               S  N  K  T  R  I  D  E  A  N  Q  R  B  T  K  M  L  SEQ ID                     __________________________________________________________________________    NO:27                                                                     

Any of the above peptides, with residue 6=L

Any of the above peptides, with residue 17 (L) deleted, with or withoutthe K/R substitutions described below

Any of the above peptides, with residue 3=K and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=K

Group 3, fair substrates

    __________________________________________________________________________    1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17                             S  N  K  T  R  I  D  E  A  N  Q  R  A  T  K  A  L  SEQ ID NO:28               S  N  K  T  R  I  D  E  A  N  N  R  A  T  K  M  L  SEQ ID NO:29               S  N  K  T  R  I  D  E  B  N  Q  R  A  T  K  M  L  SEQ ID                     __________________________________________________________________________    NO:30                                                                     

Any of the above peptides, with residue 6=L

Any of the above peptides, with residue 17 (L) deleted, with or withoutthe K/R substitutions described below

Any of the above peptides, with residue 3=K and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=K

Any of the above peptides, with one or more residues 1, 2, and/or 3deleted

Group 4, poor substrates

    __________________________________________________________________________    1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17                             S  N  K  T  R  I  D  E  A  N  Q  R  A  T  A  M  L  SEQ ID NO:31               S  N  K  T  R  I  D  E  A  N  Q  R  A  S  K  M  L  SEQ ID NO:32               S  N  K  T  R  I  D  E  A  N  A  R  A  T  K  M  L  SEQ ID NO:33               S  N  K  T  R  I  D  E  A  N  B  R  A  T  K  M  L  SEQ ID NO:34               S  N  K  T  R  I  D  E  A  A  Q  R  A  T  K  M  L  SEQ ID NO:35               S  N  K  T  A  I  D  E  A  N  Q  R  A  T  K  M  L  SEQ ID NO:36               S  N  K  T  R  I  D  E  A  N  Q  R  A  T  K  M  L  SEQ ID NO:37               S  N  K  T  R  I  D  E  A  N  Q  R  A  T  K        SEQ ID NO:38               S  N  K  T  R  I  D  E  A  N  Q  R  C  T  K  M  L  SEQ ID NO:39               S  N  K  T  R  I  D  E  A  C  Q  R  C  T  K  M  L  SEQ ID                     __________________________________________________________________________    NO:40                                                                     

Any of the above peptides, with residue 6=L

Any of the above peptides, with more than one of the above-listedsubstitutions or deletions

Any of the above peptides, with residue 3=K and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=K

Group 5, very poor substrates or non-substrates; potential inhibitors(starting points for possible anti-toxin drug development).

    __________________________________________________________________________    1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 17                                            I  D  E  A  N  Q  R  A  T  K  M  L  SEQ ID.NO:41               S  N  K  T  R  I  D  E  A  N  Q  R  L  T  K  M  L  SEQ ID NO:42               S  N  K  T  R  I  D  E  A  N  Q  K  A  T  K  M  L  SEQ ID NO:43               S  N  K  T  R  I  D  E  A  N  Q  Z  A  T  K  M  L  SEQ ID NO:44               S  N  K  T  R  I  D  E  A  N  Q  A  A  T  K  M  L  SEQ ID NO:45               S  N  K  T  R  I  D  E  A  Q  Q  R  A  T  K  M  L  SEQ ID NO:46               S  N  K  T  R  I  D  E  A  N  Q  C  A  T  K  M  L  SEQ ID NO:47               S  N  K  T  R  I  D  E  A  N  C  R  A  T  K  M  L  SEQ ID                     __________________________________________________________________________    NO:48                                                                     

Any of the above peptides, with residue 6=L

Any of the above peptides, with more than one of the above-listedsubstitutions or deletions

Any of the above peptides, with residue 3=K and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=R

Any of the above peptides, with residue 3=R and simultaneously residue15=K

It is clear from the above groupings that a very large number of changescould be made, without total elimination of substrate properties.

Peptides can be made with commercially available automated synthesizers,using reagents and protocols obtained from the manufacturers. In mostcases, solid-phase synthesis can be employed, where the C-terminalresidue is covalently attached to an insoluble resin. Subsequent aminoacids are then attached, one at a time. Amino acids can be obtained inchemically-modified ("protected") forms, designed so that they willreact with the free amino group of the preceding residue in the peptidechain, but not with themselves. Upon completion of synthesis, thepeptide is cleaved from the resin, protecting groups are removed, andthe product is purified. These preparation protocols and others are wellwithin the skill of a person in the art.

In another embodiment, the present invention relates to a kit formeasuring botulinum neurotoxin type A in a sample. The kit will containin close confinement, in a box for example, containers or vialscontaining a peptide substrate. Any of the peptide substrates can beused, preferably a peptide substrate from group 1 above. The kit willalso include a control for use as a standard for the measurement ofbotox A. The control can be a known amount of botox A, or alternatively,a known amount of the C-terminal product corresponding to the peptidesubstrate chosen, as described above. The kit should also include alabel able to detect a free amino group such that the products ofhydrolysis of the peptide substrate (and the C-terminal control, ifused) can be detected and measured. In addition, the kit can optionallyhave a container or containers with necessary buffers and cofactors forconducting the assay.

It is understood that these descriptions, examples and embodiments arefor illustrative purposes only, and that various modifications would besuggested within the spirit and purview of this application and thescope of the appended claims.

The following examples are illustrative of the practice of the inventionbut should not be read as limiting the scope thereof.

The following materials and methods were used in the examples below.

MATERIALS AND METHODS

Reagents and Chemicals

BSA, fluorescamine, and hepes were obtained from Sigma Chemical Co., St.Louis, Mo. Acetylated BSA (AcBSA) was prepared by reaction of BSA withacetic anhydride, as described (Riordan and Vallee, 1967).

Peptide synthesis

Reagents

Peptides based on the amino acid sequence of SNAP-25 (Oyler et al.,1989) were synthesized and purified as described (Schmidt and Bostian,1995, ibid.). All peptides were N-terminal acetylated, and hadcarboxamide instead of a free carboxy group at the C-terminus. Peptidenomenclature in this work differs from that in our previous report(Schmidt and Bostian, 1995). Here, peptides [1-15] , [1-16], and [1-17]are native-sequence peptides, corresponding to residues 187-201,187-202, and 187-203 of SNAP-25, respectively. For other peptides, namesindicate the amino acid normally occupying that position, then itsposition number, then its replacement. For example, in peptide K15A, thenative-sequence lysine at residue 15 is replaced by alanine.

The peptide synthesizer was a model 431A from Applied Biosystems, FosterCity, Calif. We used "Fastmoc" protocols and chemicals obtained from thesame company. Peptides were synthesized with carboxamide C-termini, byusing the Rink amide resin from Calbiochem-Novabiochem, La Jolla, Calif.N-terminal alpha amino groups were acetylated. Purification was byreverse-phase HPLC, with various gradients of 0.1% TFA and acetonitrile.Peptides were collected from the columns, lyophililzed, dissolved in 2-4ml water, and lyophilized again.

Botox A

Purified botox A was purchased from List Biological Laboratories,Campbell, Calif., and from the Food Research Institute, Madison, Wis.Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis underreducing conditions followed by staining with Coomassie Blue, eachproduct exhibited a band for the toxin heavy chain (molecular mass about100 kDa) and another for the toxin light chain (molecular mass about 50kDa). No significant amounts of other proteins were visible. Toxinconcentrations were estimated with the BCA protein assay (PierceChemical Co., Rockford, Ill.), with bovine serum albumin as thestandard. For storage, toxin was equilibrated by dialysis with 0.05Mhepes/0.15M NaCl, pH 7.3. BSA was added and toxin concentration wasadjusted, such that the solution contained 2 mg/ml BSA and 0.02 mg/mltoxin. If the toxin was to be used in the fluorescamine assay, AcBSA wassubstituted for BSA. Preparations were then stored in small aliquots(20-40 μL) at -70° C. Each aliquot was thawed and used only once.Typical assay conditions for botox A are described below. However, aswith virtually any enzymatic assay, parameters such as preincubationtime and temperature, assay time and temperature, type of buffer, bufferconcentration, pH, and concentrations of reactants and additives (botoxA, peptide substrate, DTT, ZnCl₂, BSA or AcBSA) can be altered tovarious extents, without completely eliminating botox A enzymaticactivity.

HPLC Assay of Botox A Proteolytic Activity

Peptide substrate was weighed and dissolved in a volume of 20 mM hepes,pH 7.3, such that the substrate concentration was 2 mM. An aliquot oftoxin (40 μL) was thawed and mixed with 360 μL of a solution (diluent)containing 2 mg/ml BSA or AcBSA, 10 mM DTT, 0.5 mM ZnCl₂, and 20 mMhepes, pH 7.3. Before addition of peptide substrate, this mixture waspreincubated at 37° C. for 30 min. Reaction was initiated by mixingequal volumes of peptide with preincubated botox A. Assay tubes wereincubated at 37° C. At time intervals (typically, 5-10 min apart),aliquots (usually 20 or 30 μL) were mixed with an equal volume of 2%TFA, to stop the reaction. Results were quantitated by HPLC as described(Schmidt and Bostian, 1995).

Fluorescamine Assay of Botox A Proteolytic Activity

Quantitation of proteolytic activity with fluorescamine was based on theprocedures of Galen et al. (1978). Amino groups, produced by proteolyticcleavage of peptides, are reacted with fluorescamine to give fluorescentproduct. The amount of fluorescence is proportional to the extent ofhydrolysis. Since BSA reacts strongly with fluorescamine, it wasreplaced by AcBSA in our fluorimetric assays. Use of AcBSA instead ofBSA in fluorimetric assays was reported by Wang and Liang (1994). Inthat study, the protease was porcine renin.

After assays were stopped with TFA (see section 2.3), 0.7 ml of 0.5Msodium borate/10 mM iodoacetamide, pH 9.5, was added to each aliquot,then let stand at room temperature (18-23° C.) for 30 min. This step hastwo important effects: first, the pH is made alkaline, which enhancesfluorescence yield (Galen et al., 1978), and second, iodoacetamidereacts with DTT to eliminate sulfhydryl groups. The latter must be donebecause sulfhydryl groups react very rapidly with fluorescamine andthereby prevent formation of fluorescent derivatives with amino groups.Finally, 0.1 ml of 1 mg/ml fluorescamine in dimethylformamide was added,with immediate vigorous mixing. Fluorescence was measured on aPerkin-Elmer model 650-40 fluorescence spectrophotometer. Excitation andemission wavelegths were 390 nm and 490 nm, respectively. In all assays,appropriate blanks were made by first mixing TFA with the peptidesolution, then adding botox A. Fluorescence readings from these blanks(typically, 100 or less) were subtracted from the assay values.

Calculations

Kinetic parameters of the synthetic substrates were calculated fromLineweaver-Burk plots (Segel, 1975), with peptide concentrations rangingfrom 0.02 to 3.0 mM. Results are the averages of triplicatedeterminations, ± standard deviations. Where standard deviations are notshown, the range was ±10% or less.

RESULTS AND DISCUSSION

BSA Stimulation of Botox A Proteolysis

BSA accelerated the initial rate of botox A-catalyzed hydrolysis ofpeptide [1-17](data not shown). The highest stimulation was found atabout 2 mg/ml BSA, with very similar results at 1 mg/ml. Theconcentration of BSA that produced half of the maximum effect wasapproximately 0.2 mg/ml (3 μM). In addition to peptide [1-17], similarresults were found for several other peptide substrates (not shown). Thethree different preparations of BSA, of various degrees of purity (seeMaterials and Methods), stimulated to the same extent. Dialysis of theBSA did not alter the degree of stimulation. In control experiments, BSAalone had no effect on the substrate peptides, and botox A did notcleave BSA.

Human, horse, goat, sheep, and rabbit albumins also accelerated thehydrolysis of substrate peptides by botox A, but to a lesser extent thandid BSA. Bovine gamma globulins, ovalbumin, lysozyme, ribonuclease, andgelatin had no effect, suggesting that the stimulation might be specificto serum albumins.

Kinetic analyses revealed that the increase in hydrolysis rate caused byBSA was primarily due to a tenfold increase in k_(cat), while the K_(m)decreased by a factor of three. Therefore, the catalytic step was morestrongly affected than substrate binding. Furthermore, it is clear thatthe stimulatory effect of BSA was not simply due to stabilization ofbotox A, since assay times were brief (usually ten minutes) and botox Ahas been shown to be stable for several hours under assay conditions inthe absence of BSA (Schmidt and Bostian, 1995, ibid.).

Substrate Requirements of Botox A

Analogs of peptide [1-17] were synthesized with various amino acidssubstituted for the native-sequence residues, to study the substraterequirements of botox A. In addition, peptides [1-15] and [1-16] wereincluded, to examine the effects of substrate peptide length on the rateof catalysis, in the presence of BSA. Results are summarized in Table I.

In our earlier work, no hydrolysis was detected when peptide [1-15],representing residues 187-201 of SNAP-25, was incubated with botox A(Schmidt and Bostian, 1995). However, in this study, with BSA in theassay, a very slow rate of hydrolysis was found. Nonetheless, peptide[1-15] was a relatively poor substrate for botox A, and several lines ofevidence suggest that this was due to a diminished catalytic rate,rather than a lower binding affinity for botox A. First, the estimatedk_(cat) of peptide [1-15] was only 2-3% of those for peptides [1-16] and[1-17], but the estimated K_(m) implied good binding. Second, at peptideconcentrations above 2 mM, the reaction rate decreased, relative tolower substrate concentrations, resulting in a non-linearLineweaver-Burk plot (not shown). Third, peptide [1-15] inhibitedhydrolsis of peptide [1-17] by 17%, when both were present at 1.0 mM.These findings demonstrated that peptide [1-15] can function as aninhibitor, as well as a substrate. Therefore, the low rate of peptide[1-15] hydrolysis was due to a decreased rate of catalysis, and not to alack of binding.

Extending the peptide to 16 residues by adding methionine to theC-terminus produced an excellent substrate for the proteolytic activityof botox A. The k_(cat) for peptide [1-16] is typical of substrates formany proteases and other enzymes. In contrast, the K_(m) is relativelyhigh, but similar to those reported for synthetic substrates of type Bbotulinum and tetanus neurotoxins (Cornille et al. , 1994, ibid.; Foranet al., 1994, ibid.; Shone and Roberts, 1994, ibid.). Eliminating thefirst three residues (SNK) led to a 43% reduction in hydrolysis rate(not shown). Although this 13-residue peptide was still a goodsubstrate, it was clear that of the tested peptides, the best substratehad 16 residues, with 11 on the amino-terminal side of the cleavagesite, and five on the carboxy-terminal side.

                                      TABLE I                                     __________________________________________________________________________    Kinetic Parameters of Synthetic Peptides Tested as Substrates for Botox                                 Relative                                                                           K.sub.m k.sub.cat                              Peptide                                                                              Sequence.sup.1              (mM).sup.3ate.sup.2                                                                    (sec.sup.-1)                                                                   SEQ ID NO:                       __________________________________________________________________________    [1 - 15]                                                                           S N K T R I D E A N Q R A T K                                                                      0.03 (0.9)   (1.0) SEQ ID NO:38                     [1 - 16]                                                                                      S N K T R I D E A N Q R A T K M                                                              1.6 ± 0.15.17                                                                        6 ± 4                                                                               SEQ ID NO:49                [1 - 17]                                                                                       S N K T R I D E A N Q R A T K M L                                                           1.7 ± 0.1                                                                             47 ± 2                                                                            SEQ ID NO:1                  M16A                S N K T R I D E A N Q R A T K A L                                                        1.9 ± 0.1.38                                                                          25 ± 1                                                                           SEQ ID NO:28                  M16X                S N K T R I D E A N Q R A T K X L                                                        0.58 ± 0.020                                                                       30 ± 1                                                                               SEQ ID NO:24                 K15A                S N K T R I D E A N Q R A T A M L                                                        (1.0)      (8.0)                                                                               SEQ ID NO:31                  T14S                S N K T R I D E A N Q R A S K M L                                                   0.26 (1.2)        (9.0)                                                                             SEQ ID NO:32                  T14B                S N K T R I D E A N Q R A B K M L                                                   1.20 1.3 ± 0.1                                                                           35 ± 2                                                                              SEQ ID NO:25                 A13B                S N K T R I D E A N Q R B T K M L                                                        1.8 ± 0.1.79                                                                       39 ± 2                                                                           SEQ ID NO:27                     A13L                  S N K T R I D E A N Q R L T K M L                                                       ND                 SEQ ID NO:42               R12K                  S N K T R I D E A N Q K A T K M L                                                      ND                  SEQ ID NO:43               R12Z                  S N K T R I D E A N Q Z A T K M L                                                      ND          ND02                                                                              SEQ ID NO:44                   R12A                  S N K T R I D E A N Q A A T K M L                                                 <0.02                                                                              ND      ND    SEQ ID NO:45                     Q11A                  S N K T R I D E A N A R A T K M L                                                      ND         ND 0.19                                                                                 SEQ ID NO:33              Q11B                  S N K T R I D E A N B R A T K M L                                                      0.83 ± 0.050.25                                                                    7.7 ± 0.2                                                                          SEQ ID NO:34                   Q11N                S N K T R I D E A N N R A T K M L                                                        0.75 ± 0.080.66                                                                    19 ± 3                                                                                SEQ ID NO:29                N10Q                S N K T R I D E A Q Q R A T K M L                                                        ND                  SEQ ID NO:46               N10A                S N K T R I D E A A Q R A T K M L                                                        0.60 ± 0.030.06                                                                    1.8 ± 0.1                                                                         SEQ ID NO:35                    A9B                  S N K T R I D E B N Q R A T K M L                                                       1.1 ± 0.1                                                                          11 ± 1                                                                             SEQ ID NO:30                   E8Q                    S N K T R I D Q A N Q R A T K M L                                                  2.08                                                                             0.75 ± 0.04                                                                        51 ± 3                                                                                         SEQ ID NO:18       D7N                    S N K T R I N E A N Q R A T K M L                                                  0.23                                                                             0.82 ± 0.06                                                                        5.0 ± 0.3                                                                        SEQ ID NO:50                     __________________________________________________________________________     .sup.1 Nonstandard amino acid abbreviations are: B, 2aminobutyric acid; X     2aminohexanoic acid (norleucine); and Z, 2aminopentanoic acid (norvaline)     .sup.2 Initial hydrolysis rates relative to peptide [1  17]. Peptide          concentrations were 1.0 mM.                                                   .sup.3 Numbers in parentheses wereestimated from plots of initial rates       vs. substrate concentrations (peptides [1  15] and K15A), or from the fou     lowest substrate concentrations on the LineweaverBurk plot (T14S). ND: no     determined.                                                              

Adding leucine, i.e. peptide [1-17], had little effect on kineticconstants. Nonetheless, leucine was retained in subsequent peptides,because it improved the HPLC characteristics of the C-terminalhydrolysis product.

Peptides M16A and M16X were synthesized to test the importance of havingmethionine, in particular, as residue 16. M16A was still a substrate, inspite of this relatively drastic amino acid change, although the k_(cat)decreased by almost 50%. In peptide M16X, norleucine can be consideredan analog of methionine, with a methylene group in place of the sulfur.This peptide was a good substrate, and was hydrolyzed somewhat fasterthan the native-sequence peptide at 1.0 mM, due to its lower K_(m). Insum, although residue 16 was required for efficient cleavage by botox A,it did not need to be methionine.

Peptides K15A and T14S exhibited kinetics similar to those of peptide[1-15]. A significantly lower hydrolysis rate was found, compared topeptide [1-17], and estimates of kinetic constants indicated that themain effect was on the rate of catalysis. Moreover, initial velocitiesdecreased at peptide concentrations above 2 mM, suggesting that K15A andT14S can function as inhibitors as well as substrates. Lineweaver-Burkplots were clearly non-linear and for peptide T14S the deviation fromlinearity is most noticeable at higher substrate concentrations.However, by using only the four lowest substrate concentrations, wecalculated a K_(m) of 1.2 mM and a k_(cat) of 9.0 sec⁻¹.

In peptide T14S, the side-chain methyl group of threonine is eliminated,but the hydroxyl group is retained. In T14B, the opposite situationoccurs. In contrast to T14S, peptide T14B is a good substrate for botoxA and gave typical Michaelis-Menten kinetics with constants similar tothose of the native sequence peptides. Therefore, it is clear that thehydrophobic methyl group is preferred by the enzyme at this residue,rather than the hydrophilic hydroxyl group.

Replacing alanine with 2-aminobutyric acid at residue 13 (peptide A13B)had very little effect, but substitution with the branched amino acid,leucine, caused the rate of hydrolysis to drop below the limits ofdetection. Furthermore, this peptide (A13L) inhibited hydrolysis ofpeptide [1-17] by 30%, when both were present at 1.0 mM. Preliminaryresults (not shown) indicated a Ki of about 1-2 mM for A13L. Thesefindings suggest that the binding affinity of A13L is similar to that ofpeptide [1-17], but the net rate of catalysis is greatly diminished.

Residues 11 (glutamine) and 12 (arginine) represent the P1 and P1'residues, respectively, at the site of botox A-catalyzed peptidehydrolysis (substrate terminology as defined by Schechter and Berger(1967) Biochem. Biophys. Res. Comm. 27: 157-162). Replacing the P1glutamine with alanine (Q11A), 2-aminobutyric acid (Q11B), or asparagine(Q11N) slowed but did not eliminate hydrolysis. For Q11B and Q11N, thiswas due to a decreased rate of catalysis and not to diminished binding,since Km values for these peptides were actually lower than for thenative-sequence peptides, [1-16] and [1-17]. In contrast, replacing theP1' arginine with lysine, norvaline, or alanine eliminated detectablehydrolysis. Therefore, the P1' residue is probably more important thanthe P1 for substrate recognition and cleavage by botox A.

The rate of hydrolysis was also strongly affected by amino acidsubstitutions at residue 10, the P2 site. No hydrolysis of peptide N10Qwas detected, although this is a relatively conservative change. PeptideN10A, wherein the carboxamide side chain of asparagine was eliminated,was only very slowly cleaved. For N10A, the K_(m) indicated thatsubstrate binding was somewhat stronger than in the native-sequencepeptides, but the k_(cat) was only about 4% of that for peptide [1-17].Peptide N10A inhibited hydrolysis of [1-17] by 30% when both werepresent at 1.0 mM.

In the native sequence, alanine is the P3 residue. Substitution with2-aminobutyric acid (peptide A9B) lengthened the side chain by onemethylene group. This peptide was still a good substrate at 1.0 mM, witha K_(m) slightly lower than that of peptide [1-17], but the k_(cat) wasreduced by about 75%. Although other changes to this residue have notbeen synthesized, it is likely that a relatively small, unchargedresidue is preferred at this location.

Glutamic acid at the P4 site is one of only two acidic residues in thesubstrate, compared to four basic residues. We investigated therequirement for a negative charge at this location by substituting withglutamine. Interestingly, this led to a doubling of the rate ofhydrolysis at 1.0 mM, compared to that for peptide [1-17]. Kineticanalysis revealed that this was due to a decrease in K_(m) by about 50%,while K_(cat) was unchanged. Thus, in assays at concentrations below theK_(m) of peptide [1-17], peptide E8Q appears to be the better substrate.

In contrast, eliminating the negative charge at the P5 site, bysubstituting asparagine for aspartic acid (peptide D7N), led to asubstantial decrease in catalytic rate. Although the K_(m) suggests goodbinding of D7N to botox A, the k_(cat) was only about 10% of that forpeptide [1-17]. In sum, these observations show that in the botox Asubstrate peptides, a negative charge is preferred at the P5 site, butnot at the P4 site.

Peptide Substrates for Botox A Proteolytic Activity

In earlier work, using HPLC to separate and quantitate substrate andproducts of proteolytic cleavage, we found that botox A could catalyzethe hydrolysis of a 17-residue peptide (Schmidt and Bostian, 1995).Although the alpha-amino group of this peptide was acetylated, itcontained two free amino groups, due to the presence of two lysineresidues at positions 3 and 15 in the sequence. In order to usefluorescamine detection to quantitate the enzymatic activity of botox Awithout separation of products, the two amino groups would have to beeliminated from the substrate. Otherwise, fluorescence resulting fromreaction of fluorescamine with these groups would be twice that of themaximum that could be expected from 100% cleavage of substrate,seriously compromising the sensitivity of the assay.

In the substrate peptide, replacement of lysine-15 or lysine-3 (Schmidtand Bostian, unpublished result) with alanine led to substantialdecreases (82-88%) in the rate of botox A-catalyzed hydrolysis,suggesting that positively-charged residues were required at thesesites. Therefore, peptides were synthesized with arginine as replacementfor both lysines. The modified peptides were tested as substrates forbotox A, with 1 mg/ml BSA in the assays, by comparing initial rates ofhydrolyses to that of the native-sequence peptide. Results werequantitated by the HPLC assay, and are shown in Table II.

                                      TABLE II                                    __________________________________________________________________________    Initial hydrolysis of peptide substrates                                      catalyzed by Botox A.                                                                                        Relative                                       Peptide Sequence/SEQ ID NO:                                                                                    Rate*Rate                                    __________________________________________________________________________    1  S N K T R I D E A N Q R A T K M L/1                                                                 7.6 ± 0.3                                                                        1.0                                            2  S N R T R I D E A N Q R A T R M L/2                                                                11.3 ± 0.5                                                                        1.5                                            3  S N R B R I D E A N Q R A T R M L/4                                                                15.8 ± 0.8                                                                          2.1                                          4  S N R T R I D Q A N Q R A T R M L/3                                                                    12.6 ± 0.5                                                                      1.7                                          5  S N R T R I D B A N Q R A T R M L/14                                                               12.9 ± 0.3                                                                            1.7                                        6  S N R B R I D B A N Q R A T R M L/10                                                               25.3 ± 0.7                                                                          3.3                                          7  S N R B R I D Q A N Q R A T R M L/8                                                                 14.1 ± 0.7                                                                         1.9                                          __________________________________________________________________________     *Initial rate of peptide hydrolysis, in umoles/min/mg, with 1.0 mM peptid     and 1 mg/ml BSA.                                                         

In Table II, peptide 1 is the native-sequence peptide, corresponding toresidues 187-203 of the neuronal protein, SNAP-25 (Schmidt and Bostian,1995; Oyler et al., 1989). Replacing both lysines with arginines(peptide 2) resulted in a substrate that was hydrolyzed by botox Aslightly faster than peptide 1. Thus, it was possible to eliminate thetwo free amino groups from the peptide, without adverse effects onsubstrate properties. Other sequence modifications were alsoincorporated, based on earlier work, which suggested that such changesmight result in enhanced hydrolysis rates. For example, peptide 6 washydrolyzed more than three times faster than the native-sequence peptide1.

Reaction of fluorescamine with 30 nmoles peptide 1, the amount presentin a typical assay, gave intense fluorescence, above the range of thefluorimeter (>3000). However, reaction of fluorescamine with 30 nmolespeptide 2 resulted in very low fluorescence, 37±0. Peptides 3-7 gavesimilar results.

Effects of ACBSA on Substrate Kinetic Constants

As noted above, the presence of 1 mg/ml BSA greatly enhanced the rate ofbotox A-catalyzed peptide hydrolysis. However, reaction of fluorescaminewith 30 μg BSA, the amount present in a typical assay, gave intensefluorescence, above the range of the fluorimeter (>3000). In contrast,reaction of 30 μg AcBSA gave a fluorescence of 31±1, suggesting that thelatter could replace BSA in assays, when fluorescamine quantitation wasemployed. Therefore, it was necessary to compare the effects of AcBSAwith those of BSA on the kinetic parameters of botox A-catalyzedproteolysis. Results are shown in Table III. With the exception ofpeptide 6, peptides exhibited higher initial hydrolysis rates in AcBSAthan in BSA. Values for k_(cat) were essentially unchanged (except forpeptide 3), but K_(m) values were lower, suggesting that AcBSAfacilitates binding of substrate to botox A. In sum, it was clear thatacetylation of BSA did not eliminate the positive effect of this proteinon botox A-catalyzed hydrolysis rates. Indeed, a small enhancement wasfound.

                  TABLE III                                                       ______________________________________                                        Comparison of Kinetic Parameters of Botox                                     A Substrates in BSA and AcBSA.                                                Rate.sup.1     K.sub.m (mM) K.sub.cat (sec.sup.-1)                            Peptide.sup.2                                                                       In BSA  In AcBSA In BSA                                                                              In AcBSA                                                                             In BSA                                                                              In AcBSA                            ______________________________________                                        1      8      10       1.7   0.59   47    41                                  2     11      16       1.4   0.53   68    61                                  3     16      18       0.91  0.32   75    58                                  4     13      19       0.76  0.47   64    68                                  5     13      21       0.74  0.33   69    70                                  6     24      25       0.52  0.40   89    86                                  7     14      22       ND.sup.3                                                                            0.31   ND.sup.3                                                                            73                                  ______________________________________                                         .sup.1 Initial rate of hydrolysis, in μmoles/min/mg, with 1.0 mM           peptide.                                                                      .sup.2 Peptide sequences as in Table II.                                      .sup.3 ND: not determined.                                               

Assay of Botox A Proteolytic Activity with Fluorescamine

Toxin that was stored in AcBSA, and diluent containing AcBSA, were usedin these assays. The substrate was peptide 5, at an initialconcentration of 1.0 mM. Aliquots of 20 μl were removed at timeintervals, and processed as described in the Materials and Methodssection. Results (FIG. 1) showed an increase in fluorescence with time,corresponding to botox A-catalyzed hydrolysis of the substrate(confirmed by HPLC analyses of parallel samples). Moreover, the reactionrate with 0.1 μg/ml botox A was approximately 10% of that for 1 μg/mltoxin; that is the rate was proportional to the concentration of botoxA.

The fluorescence in FIG. 1 resulted from reaction of fluorescamine withthe alpha amino group of the carboxy-terminal proteolysis product,peptide RATRML. Although peptide 5 was the substrate in this example,peptides 2 through 7 would all give the same fluorescence per mole ofcleaved substrate, because the carboxy-terminal product is the same inall.

Assay sensitivity was tested by incubating 1.0 mM peptide 7 withconcentrations of botox A ranging from 0.001 to 1 μg/ml, for 6 hr.Results are summarized in Table IV.

                  TABLE IV                                                        ______________________________________                                        Fluorescence after substrate incubation                                       with Botox A                                                                  Botox A (ug/ml)                                                                             Fluorescence (6 hrs)*                                                                       S/N**                                             ______________________________________                                        1             2378 ± 66  25.3                                              0.1           1402 ± 10  16.8                                              0.01           160 ± 16  3.0                                               0.001          11 ± 3    1.2                                               ______________________________________                                         *Blank-corrected                                                              **Signal to noise ratio, or assay fluorescence divided by the                 corresponding blank fluorescence.                                        

It appears likely that a botox A concentration of 0.001 μg/ml representsthe lower limit of sensitivity, while a more conservative estimate wouldplace this limit at 0.01 μg/ml. Nonetheless, the assay can be used overa wide range of botox A concentrations.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES:  56                                           - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Ser Asn Arg Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Ser Asn Arg Thr Arg Ile Asp Gln Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 4                                                     #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Ser Asn Arg Xaa Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 14                                                    #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Ser Asn Arg Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Xaa Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #Xaa represent Nle, or Noreleucine                                            -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Ser Asn Arg Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Xaa Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 4                                                     #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Ser Asn Arg Xaa Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 4                                                     #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Ser Asn Arg Xaa Arg Ile Asp Gln Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 4                                                     #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Ser Asn Arg Xaa Arg Ile Asp Gln Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           #8        (B) LOCATION: 4 and                                                 #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                - Ser Asn Arg Xaa Arg Ile Asp Xaa Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           #8        (B) LOCATION: 4 and                                                 #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Ser Asn Arg Xaa Arg Ile Asp Xaa Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - Ser Asn Arg Thr Arg Ile Asp Gln Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 8                                                     #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Ser Asn Arg Thr Arg Ile Asp Xaa Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 8                                                     #represent Abu, or 2-Aminobutyricaa                                                          Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                - Ser Asn Arg Thr Arg Ile Asp Xaa Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           #14       (B) LOCATION: 4 and                                                 #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #represents NorleucineFORMATION:Xaa                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Ser Asn Arg Xaa Arg Ile Asp Gln Ala Asn                                     #                 10                                                          - Gln Arg Ala Xaa Arg Xaa Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                - Thr Arg Ile Asp Glu Ala Asn Gln Arg Ala                                     #                10                                                           - Thr Arg Met                                                                         13                                                                    - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Arg Ile Asp Glu Ala Asn Gln Arg Ala Thr                                     #                10                                                           - Arg Met                                                                         12                                                                        - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - Ser Asn Lys Thr Arg Ile Asp Gln Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 4                                                     #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Ser Asn Lys Xaa Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 20:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 8                                                     #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                - Ser Asn Lys Thr Arg Ile Asp Xaa Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 21:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 4                                                     #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Ser Asn Lys Xaa Arg Ile Asp Gln Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 22:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           #8        (B) LOCATION: 4 and                                                 #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                - Ser Asn Lys Xaa Arg Ile Asp Xaa Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 23:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Ser Asn Arg Ser Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Arg Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 24:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #Xaa represents norleucineATION:                                              -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Xaa Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 25:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 14                                                    #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Xaa Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 26:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Cys Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 27:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 13                                                    #Xaa represent Abu, or 2-Aminobutyric                                                        Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Xaa Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 28:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Ala Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 29:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Asn Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 30:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 9                                                     #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                - Ser Asn Lys Thr Arg Ile Asp Glu Xaa Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 31:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Ala Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 32:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Ser Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 33:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Ala Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 34:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 11                                                    #Xaa represents Abu, or 2-Aminobutyric                                                       Acid                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Xaa Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 35:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Ala                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 36:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                - Ser Asn Lys Thr Ala Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 37:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 38:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 15 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Ala Thr Lys                                                                         15                                                            - (2) INFORMATION FOR SEQ ID NO: 39:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                 10                                                          - Gln Arg Cys Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 40:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Cys                                     #                 10                                                          - Gln Arg Cys Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 41:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 12 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - Ile Asp Glu Ala Asn Gln Arg Ala Thr Lys                                     #                10                                                           - Met Leu                                                                         12                                                                        - (2) INFORMATION FOR SEQ ID NO: 42:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Arg Leu Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 43:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Lys Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 44:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 12                                                    #Xaa represents 2-aminopentanoic acid or                                                     norvaline                                                      -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Xaa Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 45:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Ala Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 46:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Gln                                     #                10                                                           - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 47:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Cys Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 48:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Cys Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 49:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                - Ser Asn Lys Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Lys Met                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO: 50:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 17 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                - Ser Asn Lys Thr Arg Ile Asn Glu Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Lys Met Leu                                                                 15                                                            - (2) INFORMATION FOR SEQ ID NO: 51:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                - Ser Asn Arg Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Arg Met                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO: 52:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #Xaa represents Nle, or Norleucine                                            -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                - Ser Asn Arg Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Arg Xaa                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO: 53:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 4                                                     #Xaa represents Abu, or 2-Aminobutyric                                                       Acid,                                                          -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #Xaa represents Nle, or Norleucine                                            -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                - Ser Asn Arg Xaa Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Arg Xaa                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO: 54:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #Xaa represents Nle, or Norleucine                                            -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                - Ser Asn Arg Thr Arg Ile Asp Glu Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Arg Xaa                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO: 55:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                                     (B) LOCATION: 8                                                     #Xaa represents Abu, or 2-Aminobutyric                                                       Acid,                                                          -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #Xaa represents Nle, or Norleucine                                            -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                - Ser Asn Arg Thr Arg Ile Asp Xaa Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Arg Xaa                                                                     15                                                            - (2) INFORMATION FOR SEQ ID NO: 56:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 16 amino                                                #sequence (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                -     (ix) FEATURE:                                                           #8        (B) LOCATION: 4 and                                                 #Xaa represents Abu, or 2-Aminobutyric                                                       Acid,                                                          -     (ix) FEATURE:                                                                     (B) LOCATION: 16                                                    #Xaa represents Nle, or Norleucine                                            -     (ii) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                - Ser Asn Arg Xaa Arg Ile Asp Xaa Ala Asn                                     #                10                                                           - Gln Arg Ala Thr Arg Xaa                                                                     15                                                            __________________________________________________________________________

What is claimed is:
 1. A substrate peptide for determining theproteolytic acitivity of botulinum neurotoxin type A using fluorescaminedetection without separation of products, said substrate peptideconsisting of at least 16 residues of SEQ ID NO:1, wherein said peptideis modified by acetylating the N-terminal amino group and eliminatingthe negative charge at the carboxy terminus.
 2. A method for measuringamount of botulinum neurotoxin type A in a sample suspected ofcontaining botulinum neurotoxin type A, said method comprising:(i)combining said sample with a substrate peptide according to claim 1 suchthat hydrolysis of said peptide is initiated; (ii) stopping hydrolysisof said peptide at different time points; (iii) measuring amount ofhydrolysis at each time point by combining a detectable label able todetect free amino groups resulting from said hydrolysis; and (iv)comparing said measurements with amount of label produced from a knownconcentration of toxin measured under similar conditions.
 3. The methodof claim 2 wherein, said sample is selected from the group consisting ofraw, cooked and processed foods.
 4. The method of claim 2 wherein, saidsample is a clinical solution for administering to a patient.
 5. Amethod for measuring amount of botulinum neurotoxin type A in a samplesuspected of containing botulinum neurotoxin type A, said methodcomprising:(i) combining said sample with a substrate peptide accordingto claim 1 such that hydrolysis of said peptide is initiated; (ii)stopping hydrolysis of said peptide at different time points; (iii)measuring amount of hydrolysis at each time point by combining adetectable label able to detect free amino groups resulting from saidhydrolysis; and (iv) comparing said measurements with amount of labelproduced from a known concentration of toxin measured under similarconditions wherein, said substrate peptide is selected from the groupconsisting of:

    ______________________________________                                        
 1. S N R T R I D E A N Q R A T R M L (SEQ ID NO:2)                           
 2. S N R T R I D Q A N Q R A T R M L (SEQ ID NO:3)                           
 3. S N R B R I D E A N Q R A T R M L (SEQ ID NO:4)                           
 4. S N R T R I D E A N Q R A B R M L (SEQ ID NO:5)                           
 5. S N R T R I D E A N Q R A T R X L (SEQ. ID                                                                         NO:6)                                
 6. S N R B R I D E A N Q R A T R M (SEQ ID NO:7)                             
 7. S N R B R I D Q A N Q R A T R M L (SEQ ID NO:8)                           
 8. S N R B R I D Q A N Q R A T R M (SEQ ID NO:9)                             
 9. S N R B R I D B A N Q R A T R M L (SEQ ID                                                                         NO:10)                               
 10. S N R B R I D B A N Q R A T R M (SEQ ID NO:11)                           
 11. S N R T R I D Q A N Q R A T R M (SEQ ID NO:12)                           
 12. S N R T R I D B A N Q R A T R M (SEQ ID NO:13);                           and                                                                          
 13. S N R T R I D B A N Q R A T R M L (SEQ ID                                                                      NO:14).                                  ______________________________________                                    


6. The method of claim 2 wherein, said substrate peptide is S N R B R ID Q A N Q R A B R X L (SEQ ID NO:15).
 7. The method of claim 2 wherein,said substrate peptide contains modified lysine residues.
 8. The methodof claim 2 wherein, said label is fluorescamine.
 9. A method forscreening compounds suspected of inhibiting or activating botulinumneurotoxin type A, said method comprising:(i) combining said compoundwith botulinum neurotoxin type A and a substrate peptide for saidbotulinum according to claim 1 such that hydrolysis of said peptide isinitiated; (ii) measuring amount of hydrolysis by combining a detectablelabel able to detect free amino groups resulting from said hydrolysis;and (iii) detecting an inhibition or activation of botulinum neurotoxintype A by detecting a decrease or increase, respectively, in hydrolysiscompared to a similar measurement produced from a reaction without thepresence of said compound.
 10. A kit for measuring botulinum neurotoxintype A in a sample said kit containing in close confinement(i) acontainer containing a known amount of botulinum neurotoxin type A; (ii)a container containing said peptide substrate of botulinum neurotoxintype A as claimed in claim 1; (iii) a container containing a label ableto detect free amino groups resulting from the hydrolysis of saidpeptide; and (iv) a container or containers containing cofactors andbuffers necessary for conducting said assay.
 11. The substrate peptideaccording to claim 1 wherein said peptide is chosen from the groupconsisting of:

    ______________________________________                                        
 1. S N R T R I D E A N Q R A T R N L (SEQ ID NO:2)                           
 2. S N R T R I D Q A N Q R A T R M L (SEQ ID NO:3)                           
 3. S N R B R I D E A N Q R A T R M L (SEQ ID NO:4)                           
 4. S N R T R I D E A N Q R A B R M L (SEQ ID NO:5)                           
 5. S N R T R I D E A N Q R A T R X L (SEQ. ID                                                                         NO:6)                                
 6. S N R B R I D E A N Q R A T R M (SEQ ID NO:7)                             
 7. S N R B R I D Q A N Q R A T R M L (SEQ ID NO:8)                           
 8. S N R B R I D Q A N Q R A T R M (SEQ ID NO:9)                             
 9. S N R B R I D B A N Q R A T R M L (SEQ ID                                                                        NO:10)                                
 10. S N R B R I D B A N Q R A T R M (SEQ ID NO:11)                           
 11. S N R T R I D Q A N Q R A T R M (SEQ ID NO:12)                           
 12. S N R T R I D B A N Q R A T R M (SEQ ID NO:13)                           
 13. S N R T R I D B A N Q R A T R M L (SEQ ID                                                                    NO:14)                                    
 14. S N R T R I D E A N Q R A T R M (SEQ ID NO:51)                           
 15. S N R T R I D E A N Q R A T R X (SEQ ID NO:52)                           
 16. S N R B R I D E A N Q R A T R X (SEQ ID NO:53)                           
 17. S N R T R I D Q A N Q R A T R X (SEQ ID NO:54)                           
 18. S N R T R I D B A N Q R A T R X (SEQ ID NO:55);                           and                                                                          
 19. S N R R R I D B A N Q R A T R X (SEQ ID NO:56).                           ______________________________________                                    


12. The substrate peptide according to claim 1 wherein said peptide is SN R B R I D Q A N Q R A B R X L (SEQ ID NO:15).
 13. A substrate peptideaccording to claim 1 wherein lysines at positions 3 and 15 of saidpeptide are substituted with arginine.